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The Sample Preparation and Characterization (SPC) facility at EMBL Hamburg is situated next to the PETRA3 beamlines for protein crystallography and small angle scattering (SAXS) that are operated by EMBL. The facility is equipped with molecular biology and biophysical instrumentation to carry out purification and characterization of macromolecular samples. It serves a mixed community of local EMBL scientists, beamline visitors and scientists from the European Union research area. Most incoming samples are destined for high-throughput crystallization at the facility or characterization by SAXS. The facility offers standardized quality control reports on each incoming sample, including characterization by mass spectrometry and Thermofluor. Based on this report, local staff can suggest and perform optimization protocols that increase the stability of the sample. For instance, Thermofluor screens(1) were developed that probe the effect of buffers and additives that are commonly used in sample preparation. These systematic screens provide a high-throughput method to identify stabilizing conditions for sample purification, storage and structural characterization. This technique has been also a valuable asset providing a high-throughput method for assessing the crystallizability of proteins by screening for conditions which contribute to the protein sample homogeneity, stability and solubility. The home-made screens have been tested on more than 200 different protein constructs at SPC facility. The aim of the SPC facility is to integrate off-line biophysical techniques with synchrotron beamlines, to offer the European user community a full package for sample characterization. The facility is especially geared towards cell biologists with little experience in structure determination. Expert staff is available to help to plan, perform and interpret biophysical experiments. It is possible for users to book SPC equipment together with their synchrotron beamtime, for protein purification, circular dichroism and isothermal calorimetry. Funded access to the facility is currently provided by the European Community's Seventh Framework Programme Biostruct-X. For further information, please contact spc@embl-hamburg.de.
abstracts
PROXIMA 2A is a new micro-focus and energy tunable beamline dedicated to biological macromolecular crystallography at Synchrotron SOLEIL. The beamline officially opened in March 2013, and its first year of user operation has yielded excellent results. The X-ray source is a powerful in-vacuum U24 undulator coupled to a cryo-cooled Si[111] channel-cut monochromator and a pair of focussing bimorph mirrors in Kirpatrick-Baez configuration. This combination delivers a photon flux of over 10**12 ph/s into a focal spot of 10 μm × 5 μm (H×V FWHM), which is tunable over 6 – 15 keV. The supports of the optical elements have been designed to minimise the effects of vibrations and thermal dilations on the X-ray beam position, which is stable to within 5 microns over a day. The experimental station consists of a high performance micro-diffractometer, a cryostream, an area detector (ADSC Q315r), and an X-ray fluorescence detector. The X-ray energies for MAD experiments are directly calibrated on the sample. A robot equipped with a large 9 uni-puck dewar (CATS Irelec) is available to users for the automated transfer and screening of cryo-cooled samples. The users launch their experiments via an MXCuBE interface [1], which permits the centering of the sample, collecting of diffraction images, recording of X-ray spectra and the transfer of samples. The X-ray diffraction data are of an excellent quality, and the users readily exploit the micro-focused X-rays to select the best zones of their crystals. The first year of results from users has yielded a variety of success stories including novel protein structures resolved from crystals as small as 5 microns, as well as those solved by SAD & MAD methods. The future perspectives include automated helical and grid scans, in situ plate screening and multi-crystal merging techniques.
